STRICT CONTROL OF METACASPASE ACTIVITY IN TRYPANOSOMES: A MATTER OF LIFE OR DEATH

BOUVIER, LEÓN ALBERTO; NIEMIROWICZ, GABRIELA TERESA; SALAS SURDUY, EMIR; PEREZ, BRIAN; CAZZULO, JUAN JOSÉ; ALVAREZ, VANINA EDER

Caxambu-Minas Gerais

Congreso; XXXII Annual Meeting of the Brazilian Society of Protozoology - XLIII Annual Meeting on Basic Research in Chagas´ Disease; 2016

Apoptosis represents a form of programmed cell death that is critical for the development and homeostasis of multicellular organisms. In unicellular organisms, however, its existence as well as the possible role are still controversial. Despite the presence of morphological characteristics compatible with apoptosis, caspase orthologs are absent in the genomes of these organisms. About a decade ago, sequences with certain degree of similarity to those of caspases (including the conservation of the Cys-His catalytic dyad and a predicted common secondary structure) were identified in the genomes of plants, fungi and protozoans, and they were grouped into a new subfamily of peptidases named metacaspases. The first years of research in the field of metacaspases were marked by vast contradictions, mainly due to different opinions about the biochemical and functional relationship between metacaspases and caspases. However, it is clear now that metacaspases possess a completely different substrate specificity compared to that of caspases. While caspases cleave peptides and proteins after aspartic acid residues, all metacaspases studied so far have specificity towards basic (Arg/Lys) aminoacids. Another important difference among caspases and metacaspases is that the latter are absolutely dependent on calcium for activity, a property that has never been described for a caspase to date. In spite of this, they seem to share with caspases some functions, including the regulation of cell cycle and death although these observations could be related to the recently reported role in proteostasis through the removal of protein aggregates. However, since natural proteolytic substrates have scarcely been identified to date, the underlying signaling mechanisms remain an enigma. Here, I will present our current work on Trypanosoma cruzi metacaspases. To start unveiling the molecular pathways modulated by these enzymes we assessed if metacaspase interactors (identified by mass spectrometry as pulled-down proteins from transgenic parasites expressing flag-tagged metacaspases using anti-Flag agarose resin) could also be substrates of these enzymes. Using a dual-vector E. coli system we evaluated proteolytic processing when coexpressing the potential substrate with the active peptidase and by this method we identified the proteasome adaptor protein Ddi1. The cleavage site, determined by N-terminal Edman sequencing of fragments produced in vitro with both recombinant purified proteins, presented an Arg residue upstream the hydrolyzed peptide bond matching perfectly the known metacaspase specificity. Moreover, replacement of this residue by Ala completely prevented cleavage. Similar results were obtained for T. brucei and budding yeast metacaspase orthologs on their respective substrates. Interestingly, in each case cleavage occurs at a linker region that connects different domains. The in vivo proteolytic event and its consequences are currently being studied.